Sema3e/Plexin D1 Modulates Immunological Synapse and Migration of Thymocytes by Rap1 Inhibition

Abstract

Regulation of thymocyte trafficking plays an important role during thymic selection, but our understanding of the molecular mechanisms underlying these processes is limited. In this study, we demonstrated that class III semaphorin E (sema3e), a guidance molecule during neural and vascular development, directly inhibited Rap1 activation and LFA-1–dependent adhesion through the GTPase-activating protein activity of plexin D1. Sema3e inhibited Rap1 activation of thymocytes in response to chemokines and TCR stimulation, LFA-mediated adhesion, and T cell–APC interactions. Immunological synapse (IS) formation in mature thymocytes on supported lipid bilayers was also attenuated by sema3e. Impaired IS formation was associated with reduced Rap1 activation on the contact surface and cell periphery. Moreover, a significant increase of CD4+ thymocytes was detected in the medulla of mice with T cell lineage–specific deletion of plexin D1. Two-photon live imaging of thymic explants and slices revealed enhanced Rap1 activation and migration of CD69+ double-positive and single-positive cells with plexin D1 deficiency. Our results demonstrate that sema3e/plexin D1 modulates IS formation and Ag-scanning activities of thymocytes within thymic tissues.

NDR1-Dependent Regulation of Kindlin-3 Controls High-Affinity LFA-1 Binding and Immune Synapse Organization

“…Indeed, Rap1 signaling to kindlin-3 was required for stabilization of LFA-1/ICAM-1 binding, which is in agreement with the role of kindlin-3 in postadhesion events (22, 23). In line with this notion, the inhibition of Rap1 signaling led to breakdown of IS formation in mature thymocytes (46). The observation that LFA-1 activation by Mn 2+ failed to rescue IS formation in Rap1-deficient T cells further highlights the requirements for the formation of Rap1 signalosome at the inner pSMAC region to sustain LFA-1 binding to ICAM-1 and mature IS formation.…”

“…We used Rap1b f/f and Rap1a −/− ; Rap1b f/f mice that were crossed with OT-II mice expressing cre recombinase driven by a CD4 promoter to inactivate the Rap1 gene in T cells (46). In Rap1b f/f ; CD4-cre ; OT-II mice, total Rap1 protein levels were reduced to less than 10% of wild-type OT-II T cell levels (46).…”

“…We used Rap1b f/f and Rap1a −/− ; Rap1b f/f mice that were crossed with OT-II mice expressing cre recombinase driven by a CD4 promoter to inactivate the Rap1 gene in T cells (46). In Rap1b f/f ; CD4-cre ; OT-II mice, total Rap1 protein levels were reduced to less than 10% of wild-type OT-II T cell levels (46). Compared to naive wild-type OT-II T cells, naive Rap1b f/f ; CD4-cre ; OT-II T cells ( Rap1b −/− ; Rap1b knockout [KO]) exhibited immature IS with poor ICAM-1 ring formation and small central pMHC clusters (Fig.…”

“…Rap1a -null/ Rap1b fl/fl / CD4-Cre mice, Mst1 fl/fl mice, and Mst2 -null mice on the C57BL/6 background were generated as previously described (28, 46, 63). OT-II mice expressing a TCR specific for OVA 323–339 bound to I-A b , αL -null mice, and β2 -null mice were obtained from Jackson Laboratories.…”

“…For live-cell imaging, 5 × 10 5 freshly isolated naive T cells (CD62L hi CD44 lo CD4 + ) from OT-II wild-type and mutant mice were suspended in phenol red-free RPMI supplemented with 4% fetal calf serum (FCS) and 10 mM HEPES (pH 7.5) before being applied onto lipid bilayers presenting ICAM-1 and pMHC as described above (32, 46). The cells were incubated on the lipid bilayers, and single-molecule video rate imaging was performed at a 33-ms interval using an iXonUltra electron-multiplying charge-coupled-device camera (Andor) fitted with a 100× objective lens (numeric aperture [NA], 1.45) and Solis software.…”